Compositions and methods for producing gamma-carboxylated proteins

ABSTRACT

The present invention relates a host cell comprising an expression vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences and a nucleic acid molecule encoding a vitamin K epoxido reductase and associated expression control sequences and a nucleic acid molecule encoding a γ-glutamyl carboxylase and associated control sequences. The invention further relates to a method of producing a protein requiring gamma-carboxylation in high yields.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. §371 ofPCT International Application No. PCT/SE2006/000426, filed Apr. 10,2006, which claims priority to Swedish Application Serial No. 0500831-3,filed Apr. 13, 2005.

TECHNICAL FIELD

The present invention relates a host cell comprising an expressionvector comprising a nucleic acid molecule encoding a protein requiringgamma-carboxylation and associated expression control sequences and anucleic acid molecule encoding a vitamin K epoxido reductase andassociated expression control sequences, and a γ-glutamyl carboxylaseand associated control sequences. The invention further relates to amethod of producing a protein requiring gamma-carboxylation in highyields.

BACKGROUND TO THE INVENTION

Bleeding is a common clinical problem. It is a consequence of disease,trauma, surgery and medicinal treatment. It is imperative tomechanically stop the bleeding. This may be difficult or even impossibledue to the location of the bleeding or because it diffuses from many(small) vessels. Patients who are bleeding may thus require treatmentwith agents that support haemostasis. This may be blood-derived products(haemotherapy), agents that cause the release of endogenous haemostaticagents, recombinant coagulation factors (F), or agents that delay thedissolution of blood clots.

The first line treatment among the blood derived products, oftenobtained from the local hospital, are whole blood for volumesubstitution and support of haemostasis, packed red cells for theimprovement of oxygen transporting capacity, platelet concentrates toraise the number of platelets (if low or defective) and fresh frozenplasma for support of the haemostasis (blood coagulation and plateletaggregation). Second line plasma derived products that supporthaemostasis are plasma cryoprecipitate, prothrombin complexconcentrates, activated prothrombin complex concentrates and purifiedcoagulation factors. Several coagulation factors are today available ashuman recombinant proteins, inactive (coagulation factors VIII and IX)and activated (coagulation factor VIIa).

Haemophilia is an inherited or acquired bleeding disorder with eitherabnormal or deficient coagulation factor or antibodies directed towardsa coagulation factor which inhibits the procoagulant function. The mostcommon haemophilias are haemophilia A (lack coagulation factor VIII) andhaemophilia B (factor IX). The purified or recombinant singlecoagulation factors are the main treatment of patients with haemophilia.Patients with inhibitory antibodies posses a treatment problem as theymay also neutralise the coagulation factor that is administered to thepatient.

The active form of Protein C (APC) is an inhibitor of plasma coagulationby degradation of the activated coagulation factors Va and VIIIa.Recombinant APC has been shown to be an effective treatment of undueplasma coagulation in patients with sepsis.

Coagulation factors for therapeutic use can be obtained from humanplasma, although the purification process is not simple and requiresmany steps of which several aim at eliminating contaminating viruses.But even with extensive safety measures and testing of blood-derivedproducts, contamination with infectious viruses or prions cannot beruled out. Because of this risk it is highly desirable to produce humantherapeutic proteins from recombinant cells grown in media withoutanimal derived components. This is not always straightforward as manyproteins require a mammalian host to be produced in a fully functionalform, i.e. be correctly post-translationally modified. Among thecoagulation factors commercially produced in recombinant cells are FVII(NovoSeven), FVIII (Kogenate, Recombinate, Refacto) and FIX (BeneFix)(Roddie and Ludlam. Blood Rev. 11:169-177, 1997) and Active Protein C(Xigris). One of the major obstacles in obtaining large amounts of fullyfunctional recombinant human coagulation factors lies in the Gla-domainpresent in FII, FVII, FIX, FX, Protein S and Protein C. This domaincontains glutamic acid residues that are post-translationally modifiedby addition of carboxyl groups. The production of these factors arehampered by the fact that over-expression of them result inunder-carboxylated, and hence inactive, protein. The Gla modificationsare a result of the action of a vitamin K-dependent enzyme calledγ-glutamyl carboxylase (GGCX). This enzyme has been extensively studiedby many scientists, particularly those involved in coagulation factorresearch (WO-A-8803926; Wu et al. Science 254(5038):1634-1636, 1991;Rehemtulla et al, Proc Natl Acad Sci USA 90:4611-4615, 1993; Stanley J.Biol. Chem. 274(24):16940-16944, 1999; Vo et al., FEBS letters445:256-260, 1999; Begley et al, The Journal of Biological Chemistry275(46):36245-36249, 2000; Walker et al., The Journal of BiologicalChemistry 276(11):7769-7774, 2001; Bandyopadhyay, et al. Proc Natl AcadSci USA 99(3):1264-1269, 2002; Czerwiec et al., Eur J Biochem269:6162-6172, 2002; Hallgren et al, Biochemistry41(50):15045-15055,2002; Harvey et al., The Journal of BiologicalChemistry 278(10):8363-8369, 2003). Attempts to co-express GGCX withcoagulation factor FIX has been tried by at least two scientific groupsbut were not successful (Rehemtulla, et al. 1993, ibid; Hallgren et al.2002, ibid). Considering the large interest in GGCX enzymes, it may beassumed that many more trials have failed and thus have not beenreported. GGCX requires reduced vitamin K as a cofactor. The reducedvitamin K is by GGCX converted to vitamin K epoxide, which is recycledto reduced vitamin K by Vitamin K epoxidoreductase (VKOR). Thus forefficient vitamin K dependent carboxylation of proteins two enzymes arerequired, GGCX and VKOR. Cloning and identification of VKOR was reported2004 (Li et al, Nature 427:541-543, 2004, Rost et al., Nature427:537-541, 2004). The VKOR protein is a 163 amino acid polypeptidewith at least one predicted transmembrane region. From recombinant cellsexpressing VKOR activity is localized to the microsomal subcellularfraction.

For human FII (prothrombin) at least 8 out of 10 Glu residues have to becorrectly modified in order to obtain fully functional prothrombin(Malhotra, et al., J. Biol. Chem. 260:279-287, 1985; Seegers and WalzProthrombin and other vitamin K proteins', CRC Press, 1986). Similarity,human coagulation factor IX clotting activity require γ-carboxylation ofat lest 10 out of 12 glutamic residues in the Gla-domain (White et al,Thromb. Haemost. 78:261-265, 1997). Extensive efforts to obtain highproduction levels of rhFII have been made using several differentsystems such as CHO cells, BHK cells, 293cells and vaccinia virusexpression systems, but have all failed or resulted in anunder-carboxylated product and thus functionally inactive prothrombin(Jørgensen et al., J. Biol. Chem. 262:6729-6734, 1987; Russo et al.,Biotechnol Appl Biochem 14(2):222-233, 1991; Fischer et al, J Biotechnol38(2):129-136, 1995; Herlitschka et al. Protein Expr. Purif.8(3):358-364, 1996; Russo et al, Protein Expr. Purif. 10:214-225, 1997;Vo et al. 1999, ibid; Wu and Suttie Thromb Res 96(2):91-98, 1999).Earlier reported productivities for carboxylated recombinant humanprothrombin are low; 20 mg/L for mutant prothrombin (Côte et al., J.Biol. Chem 269:11374-11380, 1994), 0.55 mg/L for human prothrombinexpressed in CHO cells (fully carboxylated, Jøergensen et al. 1987,ibid), 25 mg/L in CHO cells (degree of carboxylation not shown, Russo etal. 1997, ibid).

As far as known co-expression of a protein requiring γ-carboxylation andVKOR has not been reported earlier.

WO 92/19636 discloses the cloning and sequence identification of a humanand bovine vitamin K dependent carboxylase. The application suggestsco-expressing the vitamin K dependent carboxylase and a vitamin Kdependent protein in a suitable host cell in order to prepare thevitamin K dependent protein. No co-expression of the carboxylase andvitamin K dependent protein is exemplified.

WO 92/19636 discloses the cloning and sequence identification of a humanand bovine vitamin K dependent carboxylase. The application suggestsco-expressing the vitamin K dependent carboxylase (GGCX) and a vitamin Kdependent protein in a suitable host cell in order to prepare thevitamin K dependent protein. No co-expression of the carboxylase andvitamin K dependent protein is exemplified.

WO 2005/038019 claims a method of increasing the overall productivity ofγ-carboxylated protein by a controlled co-expression of γ-carboxylatedprotein and GGCX. The invention is exemplified with improvedproductivity of coagulation factors II and FIX.

WO 2005/030039 suggests co-expression of vitamin K dependent proteinswith Vitamin K epoxide reductase (VKOR) in order to improveγ-carboxylation. However, no such co-expression expression isexemplified.

Co-expression of coagulation factor X (FX) and VKOR has been shown toimprove the share of γ-carboxylated protein by Sun et al. (Blood 106:3811-3815, 2005). Wajih et al. (JBC 280:31603-31607, 2005) has inaddition demonstrated improved share of γ-carboxylated coagulationfactor IX (FIX) by co-expression with VKOR. Both publications reportedthat VKOR incresed the share of γ-carboxylated protein but VKORco-expression did not improve the overall productivity of coagulationfactor.

There is a need for improved methods to produce activated blood clottingfactors in high yields. The present invention sets out to address thisneed.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a hostcell comprising an expression vector comprising a nucleic acid moleculeencoding a protein requiring gamma-carboxylation and associatedexpression control sequences, and an expression vector comprising anucleic acid molecule encoding a vitamin K epoxidoreductase andassociated expression control sequences, wherein the host cell furthercomprises a nucleic acid molecule encoding a γ-glutamyl carboxylase andassociated expression control sequences.

In another aspect, a cell is provided which is engineered to express (i)a protein which requires gamma-carboxylation, and (ii) vitamin Kepoxidoreductase, wherein the proteins (i) and (ii) are expressed in aratio between 10:1 and 500:1.

According to a further aspect a genetically modified eukaryotic hostcell is provided comprising: (i) a polynucleotide encoding vitamin Kepoxidoreductase protein wherein said vitamin K epoxidoreductase proteinencoding sequence is operably linked to expression control sequencespermitting expression of vitamin K epoxidoreductase protein by saidcell; (ii) a polynucleotide encoding a protein requiring carboxylationby the γ-glutamyl carboxylase protein operably linked to expressioncontrol sequences permitting expression of said protein requiringcarboxylation by said cell, and (iii) a polynucleotide encodinggamma-glutamyl carboxylase

According to yet another aspect a vector is provided comprising anucleic acid molecule encoding a protein requiring gamma-carboxylationand associated expression control sequences and a nucleic acid moleculeencoding a vitamin K epoxidoreductase and associated expression controlsequences.

According to another aspect a method is provided for producinggamma-carboxylated protein comprising:(i) culturing a cell expressing arecombinant protein which requires gamma-carboxylation, vitamin Kepoxidoreductase and a γ-glutamyl carboxylase and (ii) isolatinggamma-carboxylated protein.

According to another aspect a method is provided of producing apharmaceutical composition suitable for inducing blood clotting orpromoting increased or decreased coagulation, comprising purifyingactive carboxylated protein produced according to the above methods andadmixing the purified carboxylated protein with one or morepharmaceutically acceptable carriers or excipients.

According to a further aspect a method is provided of promotingincreased or decreased coagulation in a subject comprising administeringa pharmacologically effective amount of an isolated gamma-carboxylatedprotein obtained by the above methods to a patient in need thereof.

The protein requiring gamma-carboxylation produced by the methods of thepresent invention can be used in haemostatic or antithrombothic therapy.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a plasmid map of F10NopA (factor X+GGCX) co-expressionvector and a plasmid map of VKORzeo (VKOR) expression vector.

FIG. 2 shows plasmid maps of vectors used for co-expression of FII, GGCXand VKOR

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided a hostcell comprising an expression vector comprising a nucleic acid moleculeencoding a protein requiring gamma-carboxylation and associatedexpression control sequences, and an expression vector comprising anucleic acid molecule encoding a vitamin K epoxidoreductase andassociated expression control sequences, wherein the host cell furthercomprises a nucleic acid molecule encoding a γ-glutamyl carboxylase andassociated expression control sequences.

In one embodiment said nucleic acid molecule encoding a proteinrequiring gamma-carboxylation and associated expression controlsequences comprises a first promoter, and said nucleic acid moleculeencoding a vitamin K epoxidoreductase and associated expression controlsequences comprises a second promoter. In another embodiment the firstpromoter is sufficiently stronger than the second promoter so that theprotein requiring gamma-carboxylation and the vitamin K epoxidoreductaseare expressed in a ratio of at least 10:1. In another embodiment thefirst promoter is sufficiently stronger than the second promoter so thatthe protein requiring gamma-carboxylation and the vitamin Kepoxidoreductase are expressed in a ratio of at least 5:1.

In another embodiment the cell further comprises a nucleic acid moleculeencoding a γ-glutamyl carboxylase and associated expression controlsequences. In one embodiment, the nucleic acid molecule encoding aγ-glutamyl carboxylase and associated expression control sequencesfurther comprises a third promoter, wherein the first promoter issufficiently stronger than the third promoter so that the proteinrequiring gamma-carboxylation and the γ-glutamyl carboxylase areexpressed in a ratio of at least 10:1. In another embodiment the firstpromoter is sufficiently stronger than the second promoter so that theprotein requiring gamma-carboxylation and the vitamin K epoxidoreductaseare expressed in a ratio of at least 5:1.

The first promoter can be human cytomegalovirus (hCMV) immediate-earlypromoter and the second and third promoter can be SV40 early promoter.

In one particular embodiment, both the nucleic acid molecule encodingthe protein requiring gamma-carboxylation and associated expressioncontrol sequences, and the nucleic acid molecule encoding the Vitamin Kepoxidoreductase, and optionally the γ-glutamyl carboxylase, andassociated expression control sequences are located on the sameexpression vector. In another embodiment these two or optionally threenucleic acid molecules are located on two or more separate expressionvectors.

In another aspect a cell is provided which is engineered to express (i)a protein which requires gamma-carboxylation, and (ii) vitamin Kepoxidoreductase, wherein the proteins (i) and (ii) are expressed in aratio between 10:1 and 500:1. In another embodiment, the proteins (i)and (ii) are expressed in a ratio between 5:1 and 500:1

The protein which requires gamma-carboxylation is selected from thegroup consisting of: coagulation factor VII, coagulation FVII,coagulation factor IX, coagulation FIX, prothrombin, coagulation factorII, coagulation FII, coagulation factor X, coagulation FX, and theiractivated forms FVIIa, FIXa, FXa, Protein C, Protein S, Protein Z, BoneGla protein, Matrix Gla protein, Growth arrest-specific protein 6, snakevenom proteases similar to coagulation factors such as Factor X-likesnake venom proteases, and Acanthophiinae FXa-like protein.

In one embodiment, the protein which requires gamma-carboxylation is avitamin K dependent coagulation factor. In another embodiment, theprotein which requires gamma-carboxylation is Factor FX. In a thirdembodiment, the protein which requires gamma-carboxylation isprothrombin. In a forth embodiment, the protein which requiresgamma-carboxylation is Factor X. In a fifth embodiment, the proteinwhich requires gamma-carboxylation is factor VII.

The protein which requires gamma-carboxylation is preferably a humanprotein but all eukaryotic proteins is encompassed by the invention.Vitamin K epoxidoreductase is preferably a human protein but alleukaryotic Vitamin K epoxidoreductases can be used in the presentinvention, γ-glutamyl carboxylase is preferably a human protein but alleukaryotic γ-glutamyl carboxylases can be used in the present invention.

According to a further aspect a genetically modified eukaryotic hostcell is provided comprising:

-   (i) a polynucleotide encoding vitamin K epoxidoreductase protein    wherein said vitamin K epoxidoreductase protein encoding sequence is    operably linked to expression control sequences permitting    expression of vitamin K epoxidoreductase protein by said cell; and-   (ii) a polynucleotide encoding a protein requiring carboxylation by    the γ-glutamyl carboxylase protein operably linked to expression    control sequences permitting expression of said protein requiring    carboxylation by said cell.-   (iii) a polynucleotide encoding gamma-glutamyl carboxylase wherein    said gamma-glutamyl carboxylase protein encoding sequence is    operably linked to expression control sequences permitting    expression of gamma-glutamyl carboxylase protein by said cell

In one embodiment, the cell is capable of expressing the vitamin Kepoxidoreductase protein and the protein requiring carboxylation in theratio of at least 1:10. In another embodiment, said ratio is at least1:5.

The host cell is preferably a eukaryotic cell. Typical host cellsinclude, but are not limited to insect cells, yeast cells, and mammaliancells. Mammalian cells are particularly preferred. Suitable mammaliancells lines include, but are not limited to, CHO, HEK, NS0, 293, PerC.6, BHK and COS cells, and derivatives thereof. In one embodiment thehost cell is the mammalian cell line CHO-S.

According to yet another aspect a vector is provided comprising anucleic acid molecule encoding a protein requiring gamma-carboxylationand associated expression control sequences and a nucleic acid moleculeencoding a vitamin K epoxidoreductase and associated expression controlsequences. In one embodiment the nucleic acid molecule encodes a proteinrequiring gamma-carboxylation and associated expression controlsequences comprises a first promoter, and the nucleic acid moleculeencoding a vitamin K epoxidoreductase and associated expression controlsequences comprises a second promoter. The first promoter can besufficiently stronger than the second promoter so that the proteinrequiring gamma-carboxylation and the vitamin K epoxidoreductase areexpressed in a ratio of at least 10:1. In another embodiment this ratiois 5:1. The vector could also comprise a nucleic acid molecule encodinga γ-glutamyl carboxylase and associated expression control sequences.Said nucleic acid molecule encoding a γ-glutamyl carboxylase andassociated expression control sequences could comprise a third promoter,wherein the first promoter is sufficiently stronger than the thirdpromoter so that the protein requiring gamma-carboxylation andγ-glutamyl carboxylase are expressed in a ratio of at least 10:1. Inanother embodiment this ratio is 5:1. The protein which requiresgamma-carboxylation can be selected from the group consisting of:coagulation factor VII, coagulation FVII, coagulation factor IX,coagulation FIX, prothrombin, coagulation factor II, coagulation FII,coagulation factor X, coagulation FX, and their activated forms FVIIa,FIXa, Fxa, snake venom proteases similar to coagulation factors such asFactor X-like snake venom proteases and Acanthophiinae FXa-like protein,Protein C, Protein S, Protein Z, Bone Gla protein, Matrix Gla protein,Growth arrest-specific protein 6.

According to another aspect a method is provided for producinggamma-carboxylated protein comprising: (i) culturing a cell expressing arecombinant protein which requires gamma-carboxylation, vitamin Kepoxidoreductase and a γ-glutamyl carboxylase and (ii) isolatinggamma-carboxylated protein.

Said cell expresses the protein which requires gamma-carboxylation andvitamin K epoxidoreductase in a ratio of at least 10:1, under conditionssuitable for expression of both proteins.

The vitamin K dependent coagulation factors (FII, FVII, FIX, FX andtheir activated forms FIIa or thrombin, FVIIa, FIXa, FXa) produced bythe present method of co-expression with VKOR alone or in combinationwith GGCX can be expected to be useful in the prevention and treatmentof bleeding following trauma, surgery or diseases of the liver, kidneys,platelets or blood coagulation factors (haemophilia). Likewise thecoagulation factor Protein C and its activated form APC can be expectedto be useful in the prevention and treatment of disorders of increasedcoagulation with or without decreased levels of Protein C. The method isalso applicable to other proteins that require post-translationalcarboxylation.

The present invention will be applicable to improve the productivity ofany protein that is dependent on γ-carboxylation, such proteins include,but are not limited to: prothrombin, coagulation factor II (FII),coagulation factor VII (FVII), coagulation factor IX (FIX), coagulationfactor X (FX), Protein C, Protein S, Protein Z, Bone Gla protein (alsoknown as: BGP or osteocalcin), Matrix Gla protein (MGP), proline richGla polypeptide 1(PRRG1), proline rich Gla polypeptide 2 (PRRG2), Growtharrest-specific protein 6 (Gas 6). Other suitable proteins are: FXa-likeprotein in venom of elapid snake (subfamily Acanthophiinae) and conesnail venom (Conus textile).

Each of these proteins, including their nucleic acid and amino acidsequences, are well known. Table 1 identifies representative sequencesof wild-type and mutant forms of the various proteins that can be usedin the present invention.

TABLE 1 CDNA SPLICE VARIANTS GENE EMBL DESCRIPTION EMBL ACC# (PROTEIN)MUTATIONS ACC# Glutamate gamma BC013979 2; BC013979; AF253530 1 SNP(EMBL# U65896 carboxylase U65896); 2 SNPs (OMIM# 137167) ProthrombinV00595 1; V00595 approx. 100 SNP's AF478696 (EMBL# AF478696) Factor VIIAF466933 4; AF466933; AF272774; 21 SNPs (OMIM# J02933 AR030786; AAN60063277500) Factor IX A01819 3; A01819; A34669; 5 SNPs (EMBL# AF536327M19063 AF536327); 108 SNPs (OMIM# 306900) Factor X BC046125 4; BC040125;M57285; 118 SNPs AF503510 AR095306; AB005892 (EMBL# AF503510); 14 SNPs(OMIM# 227600) Protein C BC034377 7; AB083690; AB083693; 57 SNPs (EMBL#AF378903 I09623; S50739; S72338 AF378903); 25 SNPs (OMIM# 176860)Osteocalcin AF141310 5; AF141310; AF141310; X04143 BC033656; X04143;X51699 Matrix GLA protein BC005272 1; BC005272 Growth arrest- BC0389841; BC038984 specific 6; AXL stimulatory factor Protein Z M55670 2;AB033749; AB033749 Proline-rich Gla (G- AF009242 2; AF009242; BC030786carboxyglutamic acid) polypeptide 1 Proline-rich Gla (G- AF009243 2;AF009243; BC026032 carboxyglutamic acid) polypeptide 2 Vitamin K-BC015801 1; BC015801 approx. 100 AY308744 dependent protein SNPs (EMBL#S precursor AY308744); 8 SNPs (OMIM# 176880) Snake venom FX- AY769963Add more? like proteases AAT42490 AAT42491 AAX37260 AAX37261 AAX37262AAX37263 AAX37264 AAV34695

It will be appreciated that the invention is not restricted to aparticular protein or protein encoding sequence of one of these proteinsto be co-expressed. Moreover, and in particular with respect to bloodcoagulation factors, numerous mutant forms of the proteins have beendisclosed in the art. The present invention is equally applicable tothese mutant forms, including naturally occurring allelic variants, ofthe proteins as it is to wild-type sequence. In one embodiment theinvention can be undertaking with any wild-type protein or one with atleast 90%, preferably at least 95% sequence identity thereto.

The sequence identity between two sequences can be determined bypair-wise computer alignment analysis, using programs such as, BestFit,Gap or FrameAlign. The preferred alignment tool is BestFit. In practise,when searching for similar/identical sequences to the query search, fromwithin a sequence database, it is generally necessary to perform aninitial identification of similar sequences using suitable software suchas Blast, Blast2, NCBI Blast2, WashU Blast2, FastA, Fasta3 and PILEUP,and a scoring matrix such as Blosum 62. Such software packages endeavourto closely approximate the “gold-standard” alignment algorithm ofSmith-Waterman. Thus, the preferred software/search engine program foruse in assessing similarity, i.e., how two primary polypeptide sequencesline up is Smith-Waterman. Identity refers to direct matches, similarityallows for conservative substitutions.

The term vitamin K epoxidoreductase or “VKOR”, as used herein, refers toan enzyme that catalyses reduction of vitamin K epoxide and vitamin K toform reduced vitamin K.

Vitamin K reductases are widely distributed, and have been cloned fromseveral different species such as mouse (Mus musculus), rat (Rattusnorveigicus), chicken (Gallus gallus) and cow (Bos taurus). Homolgousproteins can be predicted from sequences from organsims of widelydispersed phylogenetic origin such as mammals, birds, amphibians, bonyfishes, flies, kinetoplastids and bacteria. Table 2 represents anon-limiting list of representative sequences of predicted proteinshomologous to human VKOR (sorted after species origin) that can be usedin the present invention.

TABLE 2 Species Data base accession #/ID Homo sapiens (man) NP_775788NP_996560 AAR28759 AAQ13668 AAQ88821 CAH10673 Bos taurus (bovine)NP_001003903 Mus musculus (mouse) NP_848715 BAB26325 NP_001001327 Rattusnorveigicus (rat) NP_976080 NP_976083 AAQ91028 Gallus gallus (chicken)NP_001001328 NP_996530 Xenopus laevis (clawed frog) AAH43742 AAH77384Xenopus tropicalis (amphibians) AAH76993 Tetraodon nigroviridis (bonyfishes) CAF98534 CAG07588 Takifugo rubripes (torafugo) AAR82913 AAR82912Anopheles gambiae (mosquito) XP_310541 EAA06271 Drosophila melanogaster(fruit fly) DAA02561 Trypanosoma brucei (protozoa) XP_340583Corynebacterium efficiens (high GC Gram+ NP_737490 bacteria)Corynebacterium glutamicum (high GC NP_600038 Gram+ bacteria)Mycobacterium leprae (high GC Gram+ NP_302145 bacteria)

The term “γ-glutamyl carboxylase” or “GGCX”, as used herein, refers to avitamin K dependent enzyme that catalyses carboxylation of glutamic acidresidues.

GGCX enzymes are widely distributed, and have been cloned from manydifferent species such as the beluga whale Delphinapterus leucas, thetoadfish Opsanus tau, chicken (Gallus gallus), hagfish (Myxineglutinosa), horseshoe crab (Limulus polyphemus), and the cone snailConus textile (Begley et al., 2000, ibid; Bandyopadhyay et al. 2002,ibid). The carboxylase from conus snail is similar to bovine carboxylaseand has been expressed in COS cells (Czerwiec et al. 2002, ibid).Additional proteins similar to GGCX can be found in insects andprokaryotes such as Anopheles gambiae, Drosophila melanogaster andLeptospira with NCBI accession numbers: gi 31217234, gi 21298685, gi24216281, gi 24197548 and (Bandyopadhyay et al., 2002, ibid),respectively. The carboxylase enzyme displays remarkable evolutionaryconservation. Several of the non-human enzymes have shown, or may bepredicted to have, activity similar to that of the human GGCX we haveused, and may therefore be used as an alternative to the human enzyme.

Table 3 identifies representative sequences of predicted proteinshomologous to human GGXC (sorted after species origin) that can be usedin the present invention.

TABLE 3 Species Data base accession #/ID Homo sapiens (man) NM_000821.2HUMGLUCARB HUMHGCA BC004422 HSU65896 AF253530.1 Papio hamadryas (redbaboon) AC116665.1 Delphinapterus leucas (white whale) AF278713 Bostaurus (bovine) NM_174066.2 BOVCARBOXG BOVBGCA Ovis aries (domesticsheep) AF312035 Rattus norvegicus (brown rat) NM_031756.1 AF065387 Musmusculus (mouse) NM_019802.1 AP087938 Opsanus tau (bony fishes)AF278714.1 Conus textile (molluscs) AY0044904.1 AP382823.2 Conusimperialis (molluscs) AF448234.1 Conus episcopatus (molluscs) AF448233.1Conus omaria (molluscs) AF448235.1 Drosophila melanogaster (fruit fly)NM_079161.2 Anopheles gambiae (mosquito) XM_316389.1 Secale cereale(monocots) SCE314767 Triticum aestivum (common wheat) AF280606.1Triticum urartu (monocots) AY245579.1 Hordeum vulgare (barley) BLYHORDCALeptospira interrogans (spirochetes) AE011514.1 Streptomyces coelicolor(high GC SCO939109 Gram+ bacteria) SCO939124 AF425987.1 Streptomyceslividans (high GC SLU22894 Gram+ bacteria) Streptomyces viginiae (highGC SVSNBDE Gram+ bacteria) Micrococcus luteus (high GC Gram+ MLSPCOPERbacteria) Chlamydomonas reinhardtii (green AF479588.1 algae)Dictyostelium discoideum (slime AC115612.2 mold) Coturnix coturnix(birds) AF364329.1 Bradyrhizobium japonicum AP005937.1(α-protoebacteria) Rhodobacter sphaeroides RSY14197 (α-proteobacteria)Sinorhizobium meliloti RME603647 (α-proteobacteria) AF119834Mesorhizobium loti AP003014.2 (α-proteobacteria) Chromobacteriumviolaceum AE016910.1 (β-proteobacteria) AE016918.1 Pseudomonasaeruginosa AE004613.1 (γ-proteobacteria) AF165882 Xanthomonas axonopodisAE011706.1 (γ-proteobacteria) Human herpesvirus 8 KSU52064 KSU75698AF305694 AF360120 AF192756

Each of the above-identified GGCX proteins can be used as thecarboxylase enzyme in the present invention.

One way to effect the co-expressed proteins is to use differentpromoters as part of the respective expression control sequences. Theart is replete with examples of different cloning vectors, promoters andother expression control sequences that are capable of expressingheterologous proteins to differing degrees or extents. Recombinantexpression technology is suitably advanced such that a person skilled inthe art of protein expression is able to select promoters and othercontrol sequences to bring about co-expression of the protein requiringcarboxylation, vitamin K epoxidoreductase and, optionally, theγ-carboxylase. The selection of which particular promoters and otherexpression control sequences to use is a matter of individual choice

In one embodiment, the control sequences associated with the proteinrequiring gamma-carboxylation comprise a strong promoter. In oneembodiment this is the human cytomegalovirus (hCMV) immediate-earlypromoter. A strong promoter is here defined as a promoter giving rise toat least 5-fold higher numbers of mRNA transcripts than a weak promoterused in the same cell under similar conditions.

In another embodiment, the control sequences associated with the vitaminK epoxido reductase, and when present the γ-glutamyl carboxylase,comprises a weak promoter. In one embodiment this is SV40 earlypromoter. In another embodiment the protein requiringgamma-carboxylation and the vitamin K epoxido reductase, and optionallythe γ-glutamyl carboxylase, are under the control of different promoterelements with the promoter controlling expression of the vitamin Kepoxido reductase, and optionally the γ-glutamyl carboxylase, beingweaker that the promoter controlling expression of the protein requiringgamma-carboxylation.

The invention has been exemplified by use of the strong CMV promoter(Boshart et al. Cell 41:521-530, 1985) to over-express Factor X and theweaker SV40 promoter (Wenger et al. Anal Biochem 221:416-418, 1994) tocontrol the expression of vitamin K epoxido reductase and optionally theGGCX expression. Other strong promoter that could be used according tothe present invention include, but are not limited to, pEF-1α [humanelongation factor-1α subunit gene) (Mizushima and Nagata, Nuc Acids Res18:5322,1990; Goldman et al., BioTechniques 21:1013-1015, 1996)], pRSV[Rous sarcoma virus (Gorman et al ., Proc Natl Acad Sci USA 79:6777-6781,1982)] and pUbC [human ubiquitin (Schorpp et al, Nuc Acids Res24:1787-1788,1996)].

The invention also extends to purified gamma carboxylated proteinproduced by the methods of the present invention and their use incoagulant therapy.

According to yet another aspect of the invention there is provided amethod of promoting increased or decreased coagulation in a subjectcomprising administering a pharmacologically effective amount of anisolated gamma-carboxylated protein obtained by the above-describedmethods to a patient in need thereof.

According to a further aspect of the invention there is provided amethod of producing a pharmaceutical composition suitable for inducingblood clotting, comprising purifying active carboxylated proteinexpressed from a host cell adapted to express a protein requiringgamma-carboxylation and γ-glutamyl carboxylase in a ratio of at least5:1 and admixing the purified carboxylated protein with one or morepharmaceutically acceptable carriers or excipients.

The compositions of the invention may be obtained by conventionalprocedures using conventional pharmaceutical excipients, well known inthe art, but will most likely be in a form suitable for injection,either parenterally or directly into the wound site.

Powders suitable for preparation of an aqueous preparation forinjection, by the addition of a suitable diluent, generally contain theactive ingredient together with suitable carriers and excipients,suspending agent and one or more stabilisers or preservatives. Thediluent may contain other suitable excipients, such as preservatives,tonicity modifiers and stabilizers.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, or a mineral oil, such as for exampleliquid paraffin or a mixture of any of these. Suitable emulsifyingagents may be, for example, naturally-occurring gums such as gum acaciaor gum tragacanth, naturally-occurring phosphatides such as soya bean,lecithin, an esters or partial esters derived from fatty acids andhexitol anhydrides (for example sorbitan monooleate) and condensationproducts of the said partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate.

The pharmaceutical compositions of the invention may also be in the formof a sterile solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, which may be formulated according toknown procedures using one or more of the appropriate dispersing orwetting agents and suspending agents, which have been mentioned above. Asterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example a solution in 1,3-butanediol.

For further information on Formulation the reader is referred to Chapter25.2 in Volume 5of Comprehensive Medicinal Chemistry (Corwin Hansch;Chairman of Editorial Board), Pergamon Press 1990; or, Volume 99 ofDrugs and the pharmaceutical sciences; Protein formulation and delivery(Eugen J. McNally, executive editor), Marcel Dekker Inc 2000.

The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. For example, a formulation intended for injection tohumans will generally contain, for example, from 0.2 mg to 6 g or from0.5 mg to 2 g of active agent compounded with an appropriate andconvenient amount of excipients which may vary from about 5 to about 98percent by weight of the total composition. Dosage unit forms willgenerally contain about 0.2 mg to about 10 g or about 1 mg to about 500mg of the active ingredient. Proteinaceous therapeutics are usuallystored frozen or freeze-dried. For further information on Routes ofAdministration and Dosage Regimes the reader is referred to Chapter 25.3in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch;Chairman of Editorial Board), Pergamon Press 1990.

The size of the dose for therapeutic or prophylactic purposes of acompound will naturally vary according to the nature and severity of theconditions, the age and sex of the animal or patient and the route ofadministration, according to well known principles of medicine. In usinga compound for therapeutic or prophylactic purposes it will generally beadministered so that a daily dose in the range, for example, 20 μg to 75mg per kg body or from 0.5 mg to 75 mg per kg body weight is received,given if required in divided doses. In general lower doses will beadministered when a parenteral route is employed. Thus, for example, forintravenous administration, a dose in the range, for example, 20 μg to30 mg per kg body weight or from 0.5 mg to 30 mg per kg body weight willgenerally be used. Similarly, for administration by inhalation, a dosein the range, for example, 20 μg to 30mg per kg or from 0.5 mg to 25 mgper kg body weight will be used. As an alternative the compound can beadministered as an infusion of 1 μg-10 mg per kilo body weight and hourduring a time period of a few hours to several days.

EXPERIMENTAL SECTION

The invention will be further described by the following non-limitingexamples.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of molecular biology and recombinant DNAtechniques within the skill of the art.

Such techniques are explained fully in the literature. See, e.g.,Sambrook et al., eds., Molecular Cloning: A Laboratory Manual (3rd ed.)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001);Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley& Sons, New York, N.Y. (2002); Glover & Hames, eds., DNA Cloning 3: APractical Approach, Vols. I, II, & III, IRL Press, Oxford (1995);Colowick & Kaplan, eds., Methods in Enzymology, Academic Press; Weir etal., eds., Handbook of Experimental Immunology, 5th ed., BlackwellScientific Publications, Ltd., Edinburgh, (1997).

Example 1

To investigate the importance of VKOR in expression of carboxylatedproteins we have expressed human coagulation factor X (FX) in CHO cells.Fully functional FX has been expressed earlier by Camire et al. 2000(Biochemistry 39:14322-14329) who obtained approximately 1 μgcarboxylated FX per million cells and day, and by Himmelspach et al 2000(Thromb Res 97:51-67) who claimed obtaining up to 25% (19.5 μg) activeFX per million cells and day using a CHO cell line that has beensubjected to DHFR amplification. Himmelspach et al. reported incompleteprocessing of recombinant FX and the maximal productivity of active FXactually shown was 5 μg/ml culture medium In both publications cellswere grown as adherent cells in serum-containing medium. Cells weregrown to desired confluence, the medium replaced with serum free mediumand incubation continued to allow accumulation of product. The amount ofproduct was then estimated from this “serum-free” medium. This cultureprocedure is not suitable for large scale protein production as thecells will only produce product for a short period. In addition theproduct will be contaminated with serum proteins such as bovine FX whichis highly undesirable as serum proteins will be difficult to remove andmay cause antibody formation if present in a product injected topatients The obtained cell lines have thus not been shown suitable forcommercial production of pharmaceutical FX.

Establisment of Stable Cell Lines Producing Recombinant Human Factor X

The FX coding sequence was PCR amplified from human liver cDNA usingprimers:

F10F1: 5′-CACACCATGGGGCGCCCACT-3′ (SEQ ID NO: 2) F10R1:5′-GAGTGGGATCTCACTTTAATGGA-3′ (SEQ ID NO: 3)

Cloning of the PCR product was first done by TA-TOPO cloning intopCDNA3.1-V5His (Invitrogen). Clones containing the correct FX sequencewere identified by DNA sequencing and by transient expression in COS-7cells. A blunt-end fragment containing the FX encoding sequence was thencloned into the EcoRV-digested and phosphatase—treated expression vectornopA. Obtained F10nopA (SEQ ID NO:6) clones were verified by DNAsequencing of the inserted sequence and by transient expression inCOS-7.

The VKOR coding sequence was PCR amplified from human liver cDNA usingprimers:

VF1: 5′-CACCATGGGCAGCACCTGGGGGA-3′ (SEQ ID NO: 4) VR1:5′-GCTCAGTGCCTCTTAGCCTT-3′ (SEQ ID NO: 5)

Cloning of the PCR product was first done by TA-TOPO cloning intopCDNA3.1-V5His (Invitrogen). Clones containing the correct VKOR encodingsequence (SEQ ID NO:1) were identified by DNA sequencing. A HindIII-NotIfragment containing the VKOR sequence was then transferred to theexpression vector pZeoSV2+ (Invitrogen) digested with the same enzymes.VKORzeo clones (SEQ ID NO:7) obtained were verified by DNA sequencing.

CHO-S cells (Invitrogen) were grown in DMEM F12 medium containingGlutamax I and 9% heat treated FBS, essentially as recommended byInvitrogen. Transfection of CHO-S was done with PvuI-digested(linearized) F10nopA, SspI-digested VKORzeo and Lipofectamine 2000essentially as recommended by Invitrogen. The DNA transfection mixcontained a 1.6-fold molar excess of F10NopA compared to VKORzeo. On theday after transfection, transfected cells were seeded in selectionmedium; growth medium plus 400 μg/ml G418, to 96-well plates. TheVKORzeo construct was thus not selected for, but was randomly integratedin the G418-resistant transfectants. Following days plates wereinspected to confirm that a suitable number of clones per well (5-10)were obtained. Six days post transfection the selection medium wasreplaced by growth medium supplemented with Vitamin K (1 μg/ml). Thenext day plates were sampled and assayed for FX activity using an assaybased on Russels' Viper Venom (RVV-X), which activates FX to FXa. FXaactivity was then measured using a chromogenic substrate (S2765,Chromogenix, Mölndal, Sweden). The RVV-X assay is eqivalent to the assayused by Himmelspach et al. for the same purpose. Wells with the highestactivity were identified and the clones contained were expanded andsubjected to limiting dilution cloning. After limiting dilution cloningand selection of the best clones, chosen clones were expanded andtransferred to growth in protein-free medium (CD-CHO supplemented asrecommended by Invitrogen plus 1 μg/ml vitamin K). Productivity ofrecombinant FX was estimated from T-flask cultures. The expression ofVKOR was assayed by ReatTime PCR analyses. It was found that allselected clones expressing fully active FX also expressed VKOR. Fromthis we conclude that co-expression of VKOR improves the expression offully active human coagulation Factor X. The obtained cell lines growwell in protein and animal component free medium and produce FX in theabsence of antibiotic selection pressure. Obtained cell lines aretherefore considered suitable for large scale protein production and arecapable of producing high amounts of active FX. The share of fullyactive FX is also significantly higher than previously reported.

Example 2

Analyses of Productivity and mRna Ratios for Co-Expression of FX, VKORand GGCX

Clones obtained in Example 1 were grown in T-flasks in protein freechemically defined CHO medium without antibiotics (Invitrogen). Sampleswere collected from 4 day cultures for preparation of cDNA and samplesfor productivity estimates were collected from cultures 5 days afterroutine split. Control samples were also prepared from the parentnon-transfected CHO-S cell line grown in the same medium and analyses ofthe control samples gave the expected results. Spinner cultures weregrown in CD-CHO with or without supplementation of animal component freeadditives. The amount of active rhFX was estimated by an assay based onRVV-X as in example 1, and a standard of serially diluted purifiedplasma derived human Factor X (Haematologic Technologies Inc., Vermont,USA). RNA was isolated with Trizol™ according to the protocol suppliedby the vendor, Invitrogen. The isolated RNA was DNaseI treated with thekit DNA-free™ from Ambion. cDNA synthesis was carried out using hexamerprimers and kit contents from Superscript™ First-Strand Synthesis Systemfor RT-PCR (Invitrogen). Primers and Vic-labeled probes for Real-TimeRT-PCR were selected using the software Primer Express™ from AppliedBiosystems.

Human γ-Carboxylase Oligonucleotides

5′ ACACCTCTGGTTCAGACCTTTCTT 3′ (SEQ ID NO: 8) Forward primer5′ AATCGCTCATGGAAAGGAGTATTT 3′ (SEQ ID NO: 9) Reverse primer5′ CAACAAAGGCTCCAGGAGATTGAACGC 3′ (SEQ ID NO: 10) ProbeHuman Factor X Oligonucleotides

Primers were manufactured by Operon/Qiagen and the probes were orderedfrom Applied Biosystems.

5′ CCGCAACAGCTGCAAGCT-3′ (SEQ ID NO: 11) Forward primer5′ TGTCGTAGCCGGCACAGA-3′ (SEQ ID NO: 12) Reverse primer5′ CAGCAGCTTCATCATCACCCAGAACATG (SEQ ED NO: 13) ProbeHuman VKOR Oligonucleotides

5′ GCTGGGCCTCTGTCCTGAT-3′ Seq(SEQ ID NO: 14) Forward primer5′ ATCCAGGCCAGGTAGACAGAAC-3′ Se(SEQ ID NO: 15) Reverse primer5′ CTGCTGAGCTCCCTGGTGTCTCTCG S(SEQ ID NO: 16) Probe

Rodent GAPDH control primers and probe were also used (AppliedBiosystems; ABI #4308318 TaqMan® Rodent GAPDH Control ReagentsProtocol)—Amplicon length 177 bp. The Real-Time RT-PCR reactions wereperformed on the 7500 Real Time PCR System, Applied Biosystems. Theexpected length of the amplified PCR products was confirmed on agarosegels.

TABLE 4 Results from Real-Time PCR analyses of FX expressing clones.Clone FX VKOR GGCX GAPDH name mRNA/cell mRNA/cell mRNA/cell mRNA/cellFX1-5 137 0.62 1 1553 FX2-5 13 0.48 0.26 2985 FX3-9 3 0.03 0.17 1891 FX6267 3 11 2289 FX17-2 319 2 37 2381

TABLE 5 Productivity estimates and mRNA ratios. Ratios are calculatedfrom data in table 4. Productivity was estimated from activity assays ofdiluted culture samples. Ratio Ratio Active FX Active FX Clone nameFX:VKOR FX:GGCX μg/ml T-flask μg/ml spinner FX1-5 221:1 137:1  0.5 Notdone FX2-5  27:1 50:1 2.4 Not done FX3-9 100:1 18:1 0.8 Not done FX6 89:1 24:1 6.9 14 FX17-2 160:1  9:1 8.7 21

The productivities listed in Table 5 are all above those previouslyobtained from non-amplified cell lines. Esimates of total FXconcentration, including inactive FX, was done using a Biacore assay andby SDS-PAGE and Western blotting.

Biacore Assay for the Estimation of the Concentration of Total rhFX

The BIAcore3000™ analytical systems, the running buffer (10 mM HEPES,0.15 M NaCl, 3.4 mM EDTA and 0.05% P20, pH 7.4), rabbit anti-mouse Fc in0.15 M NaCl (RAM Fc, lot no. 610) and the CM5 sensor chips werepurchased from Biacore AB (Uppsala, Sweden). The procedure was run at 5μl/min at 25° C. A standardised amine coupling procedure (35 μlactivation time) at 25° C. was used to covalently couple 11000 RU of thecapturing antibody RAMFc (35 μl, 30 μg/ml in 10 mM sodium-acetate, pH5.0) to channel 4 of the CMS chip. After immobilisation the surface wasregenerated with 5 μl 10 mM glycine buffer pH 1.8 and furtherequilibrated with the running buffer. With a 20 μl flow of the mouseanti-FX monoclonal IgG antibody N77121M (Biodesign, Maine, USA) (diluted1/100 in running buffer) 660 RU was captured. Binding of FX in mediumresulted in a very stable complex with negligible dissociation. For eachnew sample of FX the RAM Fc surface was reproducibly regenerated formultiple sandwich experiments. The difference in RU between channel 4with coupled RAM Fc and channel 3 with a clean surface was used toquantify the binding of 5 μl FX. A standard (2, 4,6, 8, 10, 15 and 20μg/ml in medium) of pdFX from Haematologic Technologies Inc. (Vermont,USA) was run and the difference in RU was plotted against theconcentration of phFX and the equation for one binding site was fittedto the data. The difference in RU of the unknown samples was used tocalculate the concentration of rhFX from the standard curve.

TABLE 6 Share of fully active rhFX produced. Total amount of rhFX wasestimated from spinner culture samples using a Biacore assay and amountof active rhFX was estimated by an RVV-X assay. All samples are fromspinner cultures in animal component free growth medium. Total FX μg/mlActive FX μg/ml Clone/sample (Biacore assay) (RVV-X) % active FXFX17-2/p050131 18.6 10.1 54 FX17-2/p050202 20.6 9.6 47 FX17-2/p05022511.8 12.1 103 FX17-2/sp2050309 38.3 16.3 43 FX17-2/sp1050309 25.1 13.654 FX17-2/sp2050310 39.7 21.1 53 FX17-2/sp1050310 28.1 13 46

Results in table 6 indicates that co-expression of VKOR enhances theexpression of fully active rhFX. The high share (43-103%) of fullyactive rhFX is in agreement with data from SDS-PAGE, Western blot andprotein purification.

Example 3

Co-expression of Human Prothrombin, GGCX and VKOR

To obtain a cell line capable of producing high levels of fully activehuman prothrombin (hFII) we have earlier co-expressed the vitamin Kdependent modification enzymeγ-glutamyl carboxylase (GGCX) and hFII.Using this strategy we obtained the P1E2 clone. P1E2 is a highlyproductive clone expressing rhFII, but, although expression of correctlymodified rhFII is vastly improved compared to other FII-producingclones, still only 20-60% (depending on the culture conditions) of thetotal amount of this rhFII produced is fully γ-carboxylated. In anattempt to further improve the level of fully γ-carboxylated rhFII andhence lower the production costs of rhFII, a new expression strategy wastested using vitamin K epoxide reductase (VKOR). We have cloned VKORinto two different vectors under the control of two different promoters;pCMV in the pHygro vector and pSV40 in the pZeo vector. In CHO-cells,the pCMV promoter is estimated to have an ˜6x higher promoter activitythan the pSV40 promoter. Both constructs were used in two separateco-transfections to obtain rhFII producing cell lines.

Cell Line Development and Productivity Estimates

Cell line development was initiated by cotransfecting CHO-S with the PP6construct (encoding hFII and hGGCX) (SEQ ID NO: 20) and either of theVKOR constructs (FIG. 1-2). Molar ratios used in the transfections were2:3 (PP6:VKOR). After seeding and selection of transfectants in 96-wellplates totally 5500-8500 clones per transfection were then screenedusing an ecarin based chromogenic-assay. 18 clone pools were selectedafter the initial screen. After the second screen 9 clone pools wereselected, expanded and subjected to a third screening assay. For eachtransfection the best producing clone pool was selected for limitingdilution. Six 96-well plates were seeded with 0.5 cells/well. 24 cloneswas selected and upscaled after screening. As the vectors encoding VKORhave not been selected for, Taqman analyses were done to verify VKORexpresssion. In all the three top clones selected; A3F4 (PP6/pZeoVKOR),B11E8 and B9A12 (PP6/pHygroVKOR), VKOR mRNA was detected. Four runs ofspinner experiments were done to evaluate and compare the productivityof rhFII for the PP6/VKOR clones compared to the P1E2 clone.

TABLE 7 Production of rhFII in spinner flasks. Experi- Active Share ofactive rhFII Cell Sample ment rhFII SPR (active rhFII/total line ID run(μg/mL) (pg/cell/day) rhFII in %) A3F4 050317 A 10.3 1.38 100 A3F4050415 B 27.6 1 100 A3F4 050425 C 23 2.6 100 B9A12 050317 A 10.3 0.84 68B9A12 050413 B 27.4 1 100 B9A12 050424 C 14.4 6.06 79 B11E8 050317 A 131.74 75 B11E8 050414 B 27.4 2.2 80 B11E8 050424 C 22.2 4 78 P1E2 050415B 37.6 2.1 61 P1E2 050424 C 25.4 2.5 21

The novel approach to co-express VKOR, GGCX and rhFII resulted inseveral rhFII-expressing clones producing a much higher share (60-100%)of fully active rhFII compared to the P1E2 clone (20-60%, see table 1).For two of the clones, B11E8 and B9A12 (both PP6/pHygroVKORcotransfection) a higher specific productivity rate (amount of activeprotein produced per cell and day, SPR) than the P1E2 clone was obtainedunder some culture conditions. The A3F4 clone produced 100% fullycarboxylated rhFII in all the culture experiments run. This clone hasthe highest mRNA ratio of both modification enzymes (GGCX and VKOR) toFII compared with the other two clones. However, A3F4 does not producemore fully active rhFII than the other clones.

Example 4

Improved γ-Carboxylation by Supertransfection with VKOR

In a second attempt to use VKOR for improvement of rhFII production, theP1E2 clone (example 3) was modified to co-express VKOR. The pHygroVKORconstruct (SEQ ED NO: 22) was in this case used to transfect P1E2 (seeAppendix, FIG. 1) and clones were screened for improved productivity bya prothrombinase activity assay. Totally 7000-8000 clones were screenedusing an end-point prothrombinase assay adapted to 96-well format.Sixteen clone pools were selected after the initial screen. Chosen clonepools were expanded and screened both with both ecarin andprothrombinase assay in order to estimate the share of active rhFII.After this screen 6 clone pools were selected and expanded. The threebest producing clone pools were selected for limiting dilution cloning.Twenty-eight clones originating from all three pools were selected andup-scaled after the initial prothrombinase screen. After a secondscreen, eight clones were selected and up-scaled. Taqman analyses weredone to verify VKOR expression in one clone from each cloned pool. Inall the three top clones selected (M3F6, P4A4 and O3G3), VKOR mRNA wasdetected. Three runs of shaker or spinner cultures were done to evaluatethe productivity of rhFII for the P1E2/VKOR clones compared to theparent P1E2 clone.

Taqman analyses were done to verify VKOR expression in one clone fromeach cloned pool. In all three top clones selected; M3F6, P4A4 and O3G3,VKOR mRNA was detected. Because of the selection and screeningprocedures used to obtain these clones, they are considered to expressan optimal level of VKOR expression. This optimal expression level isfurther characterized in example 5.

TABLE 8 Production of rhFII at peak productivity in spinner/shakercultures using animal component free media. SPR; Sample Viable Active/specific (Clone cell Total Active total productivity and Experimentdensities Viability rhFII rhFII rhFII rate Date) series (cells/ml) (%)mg/L mg/L (%) pg/cell/day P1E2 A 1700000 87 211.9 40 19 7.2 050622 P1E2B 3866666 93 94.7 48.4 51 1.7 050610 P1E2 C 2500000 nd 47.9 38.4 80 nd051011 M3F6 A 2550000 83 181.6 74.7 41 21.5 050622 M3F6 B 1950000 5980.2 55 69 1.8 050609 O3G3 A 3525000 >95 194.6 63.7 33 3.1 050621 O3G3 B3200000 85 128.7 68.6 53 4.2 050609 O3G3 C 6400000 nd 72.8 76.8 105 nd051010 P4A4 A 2700000 >95 176.9 34.1 19 3.3 050621 P4A4 B 2233333 81101.6 64.2 63 3.8 050610 The P1E2 parent cell line not containing theVKOR construct was grown in paralell under the same conditions as acontrol.

Results from the culture experiments showed that the amount and share ofactive rhFII during different culture conditions varied, but for mostruns the amount of fully active rhFII produced was better for the novelP1E2/VKOR clones than for the original P1E2 cell line.

Example 5

Establishment of Optimal mRNA Expression Ratios

Messenger RNA prepared from the cell lines in Example 4 and 5 wasanalysed with Real-Time PCR similarly as in Example 3. For GGCX and VKORthe same oligonucleotides as in Example 3 were used.

Oligonucleotides for prothrombin were:

5′ TGGAGGACAAAACCGAAAGAGA 3′ (SEQ ID NO: 17) Forward primer5′ CATCCGAGCCCTCCACAA 3′ (SEQ ID NO: 18) Reverse primer5′ CTCCTGGAATCCTACATCGACGGGC 3′ (SEQ ID NO: 19) Probe

TABLE 9 Analyses of mRNA ratios at peak expression of human prothrombin(rhFII) Best productivity mRNA mRNA mRNA obtained * ratio ratio ratio(active rhFII Cell line FII/GGCX FII/VKOR VKOR/GGCX mg/L) A3F4 5 13 0.427.6 B9A12 86 32 3 27.4 B11E8 29 33 0.9 27.4 M3F6 304 15 20 74.7 O3G3218 17 13 76.8 P4A4 255 128 2 64.2 P1E2 (control 221 No VKOR No VKOR48.4 without detected detected VKOR) * Measurement of productivity doneunder similar conditions in spinner or shake flasks.

Results in table 9 indicates that there is an optimal expression levelof GGCX and VKOR in relation to the γ-carboxylated protein produced.Clones M3F4, O3G3 and P4A4 were obtained by transfecting P1E2 (earlierobtained by transfection with a construct containing rhFII+GGCX) with aconstruct containing VKOR under the control of the strong CMV promoter.Screening was perfoimed with an assay specifically detecting clones withan improved productivity of fully active rhFII. Clones with an optimalexpression level of VKOR in relation to rhFII and GGCX have thus beenselected.

Messenger RNA prepared from the cell lines in Example 4 and 5 wasanalysed with Real-Time PCR similarily as in Example 3. All analysesincluded a GAPDH control reaction as in Example 3.

1. An in vitro host cell comprising: a first recombinant DNA comprisinga sequence encoding a protein requiring gamma-carboxylation operablylinked to a first expression control sequence; a second recombinant DNAcomprising a sequence encoding a vitamin K epoxidoreductase (VKOR)operably linked to a second expression control sequence; and a thirdrecombinant DNA comprising a sequence encoding a γ-glutamyl carboxylaseoperably linked to a third expression control sequence, wherein theprotein requiring gamma-carboxylation and the VKOR are expressed, in thecell in a ratio of at least 10:1.
 2. The host cell of claim 1, whereinmRNA encoding the protein requiring gamma-carboxylation and mRNAencoding a VKOR are expressed in the cell in a ratio of at least 10:1.3. The host cell of claim 1, wherein mRNA encoding the protein requiringgamma-carboxylation and mRNA encoding γ-glutamyl carboxylase areexpressed in the cell in a ratio of at least 10:1.
 4. The host cell ofclaim 1, wherein the first recombinant DNA encoding a protein requiringgamma-carboxylation and the DNA encoding a γ-glutamyl carboxylase arelocated on a single expression vector in the cell.
 5. The host cell ofclaim 1, wherein the first recombinant DNA encoding a protein requiringgamma-carboxylation, the second recombinant DNA encoding a VKOR, and theDNA encoding a γ-glutamyl carboxylase are located on a single expressionvector in the cell.
 6. The host cell of claim 1, wherein the firstexpression control sequence comprises a first promoter, the secondexpression control sequence comprises a second promoter, and theactivity of the first promoter in the host cell is greater than theactivity of the second promoter.
 7. The host cell of claim 1, whereinthe first promoter is selected from the group consisting of humancytomegalovirus (hCMV) immediate-early promoter, human elongationfactor-1 α subunit gene promoter (pEF-1α), Rous sarcoma virus promoter(pRSV), and human ubiquitin promoter (pUbC).
 8. The host cell of claim6, wherein the first promoter is hCMV immediate-early promoter, and thesecond promoter is SV40 early promoter.
 9. The host cell of claim 6,wherein the third expression control sequence comprises a thirdpromoter, and the activity of the first promoter in the host cell isgreater than the activity of the third promoter.
 10. The host cell ofclaim 1, wherein the protein requiring gamma-carboxylation is selectedfrom the group consisting of: coagulation factor VII, coagulation factorIX, prothrombin, coagulation factor X, Factor X-like snake venomproteases, Protein C, Protein S, Protein Z, osteocalcin, Matrix Glaprotein, and Growth arrest-specific protein
 6. 11. The host cell ofclaim 1, wherein the protein requiring gamma-carboxylation is a vitaminK dependent coagulation factor.
 12. The host cell of claim 1, whereinthe protein requiring gamma-carboxylation is coagulation factor IX. 13.The host cell of claim 1, wherein the protein requiringgamma-carboxylation is coagulation factor X.
 14. The host cell of claim1, wherein the protein requiring gamma-carboxylation is a Factor X-likesnake venom protease.
 15. The host cell of claim 1, wherein the proteinrequiring gamma-carboxylation is prothrombin.
 16. The host cell of claim1, wherein the protein requiring gamma-carboxylation is coagulationfactor VII.
 17. The host cell of claim 1, wherein the protein requiringgamma-carboxylation is Protein C.
 18. The host cell of claim 1, whereinthe cell is a mammalian cell.
 19. The host cell of claim 1, wherein thecell is a yeast cell or an insect cell.
 20. The host cell of claim 1,wherein the cell is a CHO cell, a HEK cell, a NS0 cell, a Per C.6 cell,a BHK cell, or a COS cell.
 21. An in vitro cell engineered to express(i) a protein that requires gamma-carboxylation, and (ii) a VKOR,wherein the cell expresses (i) and (ii) in a ratio between 10:1 and500:1.
 22. A method for producing a composition, the method comprising:(a) providing a recombinant cell comprising a first nucleic acidsequence encoding a protein requiring gamma-carboxylation operablylinked to a first expression control sequence, a second and heterologousnucleic acid sequence encoding a VKOR operably linked to a secondexpression control sequence, and a third nucleic acid sequence encodinga γ-glutamyl carboxylase operably linked to a third expression controlsequence; (b) culturing the cell in vitro under conditions suitable forexpressing each nucleic acid sequence, wherein (i) the protein requiringgamma-carboxylation and the VKOR are expressed in the cell in a ratio ofat least 10:1, and (ii) the protein requiring gamma-carboxylation iscarboxylated in the cell, thereby producing a gamma-carboxylatedprotein; and (c) isolating the gamma-carboxylated protein or anactivated form thereof.
 23. The method of claim 22, further comprising:(d) preparing a pharmaceutical composition comprising the isolatedgamma-carboxylated protein or an activated form thereof.
 24. The methodof claim 22, wherein mRNA encoding the protein requiringgamma-carboxylation and mRNA encoding VKOR are expressed in the cell ina ratio of at least 10:1.
 25. The method of claim 22, wherein mRNAencoding the protein requiring gamma-carboxylation and mRNA encodingγ-glutamyl carboxylase are expressed in the cell in a ratio of at least10:1.
 26. The method of claim 22, wherein both the first and thirdnucleic acid sequences are on the same expression vector in the cell.27. The method of claim 22, wherein the first, second and third nucleicacid sequences are on the same expression vector in the cell.
 28. Themethod of claim 22, wherein the first expression control sequencecomprises a first promoter, the second expression control sequencecomprises a second promoter, and the activity of the first promoter inthe cell is greater than the activity of the second promoter.
 29. Themethod of claim 28, wherein the first promoter is selected from thegroup consisting of human cytomegalovirus (hCMV) immediate-earlypromoter, human elongation factor-1α subunit gene promoter (pEF-1α),Rous sarcoma virus promoter (pRSV), and human ubiquitin promoter (pUbC).30. The method of claim 28, wherein the first promoter is hCMVimmediate-early promoter and the second promoter is SV40 early promoter.31. The method of claim 22, wherein the first expression controlsequence comprises a first promoter, the second expression controlsequence comprises a second promoter, the third expression controlsequence comprises a third promoter, and the activity of the firstpromoter in the cell is greater than the activity of the third promoter.32. The method of claim 31, wherein the first promoter is selected fromthe group consisting of hCMV immediate-early promoter, pEF-1α, pRSV, andpUbC.
 33. The method of claim 31, wherein the first promoter is hCMVimmediate-early promoter, and the third promoter is SV40 early promoter.34. The method of claim 31, wherein the activity of the first promoterin the recombinant cell is greater than the activity of each of thesecond and third promoters.
 35. The method of claim 34, wherein thefirst promoter is selected from the group consisting of hCMVimmediate-early promoter, pEF-1α, pRSV, and pUbC.
 36. The method ofclaim 34, wherein the first promoter is hCMV immediate-early promoter,and each of the second and third promoters is SV40 early promoter. 37.The method of claim 22, wherein the cell is a mammalian cell.
 38. Themethod of claim 22, wherein the cell is a yeast cell or an insect cell.39. The method of claim 22, wherein the cell is a CHO cell, a HEK cell,a NS0 cell, a Per C.6 cell, a BHK cell, or a COS cell.
 40. The method ofclaim 22, wherein the protein requiring gamma-carboxylation is selectedfrom the group consisting of: coagulation factor VII, coagulation factorIX, prothrombin, coagulation factor X, Factor X-like snake venomproteases, Protein C, Protein S, Protein Z, osteocalcin, Matrix Glaprotein, and Growth arrest-specific protein
 6. 41. The method of claim22, wherein the protein that requires gamma-carboxylation is a vitamin Kdependent coagulation factor.
 42. The method of claim 22, wherein theprotein that requires gamma-carboxylation is coagulation factor IX. 43.The method of claim 22, wherein the protein that requiresgamma-carboxylation is coagulation factor X.
 44. The method of claim 22,wherein the protein that requires gamma-carboxylation is a Factor X-likesnake venom protease.
 45. The method of claim 22, wherein the proteinthat requires gamma-carboxylation is prothrombin.
 46. The method ofclaim 22, wherein the protein that requires gamma-carboxylation iscoagulation factor VII.
 47. The method of claim 22, wherein the proteinthat requires gamma-carboxylation is Protein C.
 48. The host cell ofclaim 1, wherein the protein requiring gamma-carboxylation and the VKORare expressed in the cell in a ratio between 10:1 and 500:1.
 49. Themethod of claim 22, wherein the protein requiring gamma-carboxylationand the VKOR are expressed in the cell in a ratio between 10:1 and500:1.